Fertility of lateral spikelets determines the two-rowed or six-rowed spikes of barley (Hordeum vulgare L.), which results in significantly different grain yields. The change in row type from two-rowed to six-rowed shows remarkable domestication characteristics. The Qinghai-Tibet plateau has abundant resources of wild and cultivated barley, and is considered one of the centers of domestication and genetic diversity of cultivated barley. In order to obtain a primary understanding of the genetic basis of lateral spike development regulation and the domestication process in cultivated Tibetan barley, an F2 segregation population was constructed by crossing the two-rowed wild barley accession ZYM0083 with the six-rowed landraces Linzhiheiliuleng. Genetic analysis showed that the row type was controlled by a single gene. Using the specific-locus amplified fragment sequencing (SLAF-seq) technology and bulked segregant analysis (BSA), two DNA pools from 22 two-rowed spike individuals and 22 six-rowed spike individuals of the F2 population were constructed and sequenced. A total of 456 691 SLAF tags were obtained. By adopting the ED and SNP index for association analysis, three candidate regions with a 53.84-Mb interval and containing 536 genes were obtained. Four-hundred thirteen, 189, and 160 annotated genes were acquired by GO, KEGG, and COG libraries, respectively. Loci that control lateral spike development in Tibetan barley were primarily mapped by SLAF-seq, and the results presented in this study will facilitate the fine mapping and cloning of target genes.